An aspect of the present invention relates generally to the field of compounds and treatment of diseases associated with an activated inflammatory pathway or idiopathic pulmonary fibrosis (IPF).
IPF is a progressive fibrotic disease characterized by massive neovascularization and deposition of extracellular matrix into the interstitium. IPF is the most common form of interstitial lung disease with a prevalence of 50 per 100,000 cases, and it almost exclusively affects patients older than 50. Despite its unknown etiology, the downstream effectors of IPF are well-characterized. Samples from IPF patients show increased levels of transforming growth factor beta (TGFβ) across all three isoforms (Annes et al., “Making sense of latent TGFβ activation,” Journal of cell science, 116(Pt 2): 217-224 (2003)). TGFβ serves as a critical pro-inflammatory molecule via the induction of neutrophil chemotaxis, the activation of epithelial to mesenchymal transition, and the promotion of lung epithelial cell apoptosis (Kage et al., “EMT and interstitial lung disease: a mysterious relationship,” Current opinion in pulmonary medicine, 18(5): 517-523 (2012)). The TGFβ signaling pathway proceeds in part through the mothers against decapentaplegic homolog (SMAD) protein family. SMAD proteins regulate a variety of cellular processes, such as differentiation, proliferation, tumorogenesis, and immune responses (Attisano et al., “SMADS as transcriptional co-modulators,” Current opinion in cell biology, 12(2): 235-243 (2000); Yingling et al., “Development of TGF-beta signalling inhibitors for cancer therapy,” Nature reviews Drug discovery, 3(12): 1011-1022 (2004); Bonniaud et al., “TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis,” Journal of immunology, 175(8): 5390-5395 (2005)). The SMAD family is comprised of receptor-SMADs (R-SMAD), inhibitor SMADs (I-SMAD), and the common mediator SMAD (co-SMAD) (Deiynck et al., “SMAD-dependent and SMAD-independent pathways in TGF-beta family signaling,” Nature, 425(6958): 577-584 (2003)). Briefly, TGFβ signal transduction commences with the phosphorylation of R-SMADs, often SMAD2 or SMAD3, which form a trimeric structure with the co-SMAD. SMAD4, and translocate to the nucleus to bind to the SMAD binding element (SBE) in the JunB promoter to activate transcription (Jonk et al., “Identification and functional characterization of a SMAD binding element (SBE) in the JunB promoter that acts as a transforming growth factor-beta, activin, and bone morphogenetic protein-inducible enhancer,” The Journal of biological chemistry, 273(33): 21145-21152 (1998)). Therefore, TGFβ is a major pro-fibrotic growth factor through the downstream SMAD signaling pathway (Wang et al., “Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis,” The Journal of experimental medicine, 203(13): 2895-2906 (2006); Hecker et al., “NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury,” Nature medicine, 15(9): 1077-1081 (2009)).
PIAS (protein inhibitor of activated STAT) proteins are a family of proteins that are known to negatively control and regulate gene transcription and inflammatory pathways in cells (Rytinki et al., “PIAS proteins: pleiotropic interactors associated with SUMO,” Cell Mol Life Sci, 66(18): 3029-3041 (2009)). There are four characterized PIAS family members, PIAS1, PIASx (PIAS2), PIAS3, and PIASy (PIAS4), each with specificity toward different pathways (Gross et al., “Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells,” Oncogene, 20(29): 3880-3887 (2001)). Specifically, PIAS4 has been shown to suppress TGFβ signaling (Long et al., “Repression of SMAD transcriptional activity by PIASy, an inhibitor of activated STAT,” Proceedings of the National Academy of Sciences of the United States of America, 100(17): 9791-9796 (2003); Imoto et al., “Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3,” The Journal of biological chemistry, 278(36): 34253-34258 (2003)). First, TGFβ promotes PIAS4's interaction with SMAD3 and SMAD4 to form a ternary complex PIAS4 is known to possess small ubiquitin-like modifier (SUMO) E3 ligase activity within its RING-type domain (Imoto et al., “The RING domain of PIASy is involved in the suppression of bone morphogenetic protein-signaling pathway,” Biochemical and biophysical research communications, 319(1): 275-282 (2004)), so it promotes the sumoylation of SMAD3, in turn stimulating its nuclear export and inhibiting SMAD3/4 driven transcription (Imoto et al., “Sumoylation of SMAD3 stimulates its nuclear export during PIASy-mediated suppression of TGF-beta signaling,” Biochem Biophys Res Commun, 370(2): 359-365 (2008); Lee et al., “Sumoylation of SMAD4, the common Smad mediator of transforming growth factor-beta family signaling,” The Journal of biological chemistry, 278(30): 27853-27863 (2003)). Furthermore, PIAS4 directly recruits and interacts with histone deacetylase 1 (HDAC1) to repress SMAD3 driven transcriptional activation. In all, PIAS4 is an important negative regulator of TGFβ signaling.
Protein ubiquitination is the major protein processing function in cells. Ubiquitin (Ub) flags a targeted protein for degradation through the 26 s proteasome or lysosome (Tanaka et al., “c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130,” Mol Cell Biol., 28(15): 4805-4818 (2008)). Ubiquitin is conjugated to a target protein in a three-step process. First, an E1 ubiquitin-activating enzyme binds to ubiquitin via a thioester covalent bond. Then, the E1 transfers the ubiquitin to an E2 ubiquitin-conjugating enzyme. Finally, the C-terminus of Ub is attached to the ε-amino lysine (K) residue of the substrate, mediated by a ubiquitin E3 ligase. There are several families of these ubiquitin E3 ligases that include over 1,000 proteins (Jin et al., “Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging,” Nature, 447(7148): 1135-1138 (2007); Hatakeyama et al., “U box proteins as a new family of ubiquitin-protein ligases,” J Biol Chem, 276(35): 33111-33120 (2001)). Of these, the E6-AP Carboxyl Terminus (HECT) domain E3 ligase family remains poorly characterized (Rotin et al., “Physiological functions of the HECT family of ubiquitin ligases,” Nature reviews Molecular cell biology, 10(6): 398-409 (2009)). There are ˜30 HECT E3 ligases in mammalian cells, and functional data is only available for a select few including E6AP, Smurf, and NEDD4. HECT E3 ligases possess a unique feature in which they accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester bond and directly transfer the ubiquitin to the substrate. An active site within the C-terminal of the HECT domain containing a cysteine residue is required for ubiquitin-thiolester formation (Huibregtse et al., “A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase,” Proceedings of the National Academy of Sciences of the United States of America, 92(7): 2563-2567 (1995)). A recently identified member of the HECT E3 ligases, AREL1 encoded by the KIAA0317 gene, regulates the ubiquitination of the apoptosis proteins SMAC, HtrA2, and ARTS (Kim et al., “Identification of a novel anti-apoptotic E3 ubiquitin ligase that ubiquitinates antagonists of inhibitor of apoptosis proteins SMAC, HtrA2, and ARTS,” J Biol Chem, 288(17): 12014-12021 (2013)).
Thus, there remains a need in the art to discover the role of the protein encoded by KIAA0317 in various signaling pathways. Futhermore, there remains a need in the art to develop new antagonists that exert potent anti-fibrotic activity for treating idiopathic pulmonary fibrosis (IPF). The present invention satisfies these needs.